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de Oliveira Dias et al. Int J Retin Vitr (2016) 2:3 DOI 10.1186/s40942-016-0026-y International Journal of Retina and Vitreous

REVIEW Open Access Fusion proteins for treatment of retinal diseases: aflibercept, ziv‑aflibercept, and conbercept João Rafael de Oliveira Dias* , Gabriel Costa de Andrade, Eduardo Amorim Novais, Michel Eid Farah and Eduardo Büchele Rodrigues

Abstract In the last few years, monoclonal have revolutionized the treatment of retinal neovascular diseases. More recently, a different class of drugs, fusion proteins, has provided an alternative treatment strategy with pharmacologi- cal differences. In addition to commercially available aflibercept, two other drugs, ziv-aflibercept and conbercept, have been studied in antiangiogenic treatment of ocular diseases. In this scenario, a critical review of the currently available data regarding fusion proteins in ophthalmic diseases may be a timely and important contribution. Afliber- cept, previously known as VEGF Trap Eye, is a of VEGF receptors 1 and 2 and a treatment for several retinal diseases related to angiogenesis. It has firmly joined and as an important thera- peutic option in the management of neovascular AMD-, DME- and RVO-associated . Ziv-aflibercept, a systemic chemotherapeutic agent approved for the treatment of metastatic , has recently drawn attention because of its potential for intravitreal administration, since it was not associated with ERG-related signs of toxicity in an experimental study and in human case reports. Conbercept is a soluble decoy that blocks all isoforms of VEGF-A, VEGF-B, VEGF-C, and PlGF, which has a high binding affinity for VEGF and a long half-life in vitre- ous. It has been studied in a phase three and has shown efficacy and safety. This review discusses three fusion proteins that have been studied in ophthalmology, aflibercept, ziv-aflibercept and conbercept, with emphasis on their clinical application for the treatment of retinal diseases. Keywords: Fusion proteins, Aflibercept, Ziv-aflibercept, Conbercept, Vascular endothelial (VEGF), VEGF Trap Eye

Introduction angiogenesis [2]. Three VEGF receptor (VEGFR) sub- Vascular endothelial growth factor (VEGF) is a 36- to types have been identified: VEGFR 1–3, among which 46-kDa homodimeric glycoprotein that acts as an angio- VEGFR-1 binds VEGF with the highest affinity, while genic cytokine, inducing mitosis [1]. It has six members, VEGFR-2 is the most important in angiogenesis [2]. VEGF-A to -E, and (PlGF), of Many cell types in the retina produce VEGF. These which VEGF-A is the most important cytokine involved include the retinal pigment epithelium (RPE), vascular in angiogenesis. There are several isoforms of VEGF- endothelial cells, pericytes, retinal neurons, Müller cells A in humans: VEGF121, VEGF145, VEGF165, VEGF183, and astrocytes [3, 4]. VEGF is secreted by the RPE and VEGF189 and VEGF206. Of these, VEGF165 is the most retinal cells in response to hypoxia secondary to ischemic common VEGF-A isoform and the most important for retinal disorders. Upregulation of VEGF results in angio- genesis, increased vascular permeability, and the produc- tion of pro-inflammatory cytokines [5]. *Correspondence: [email protected] Over the past decade, the use of intravitreal pharma- Department of Ophthalmology, Federal University of São Paulo-Paulista Medical School, Rua Botucatu, 821, 1st Floor, São Paulo, SP 04023‑062, cotherapy to block VEGF has become common and has Brazil significantly improved visual outcomes in patients with

© 2016 de Oliveira Dias et al. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons. org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. de Oliveira Dias et al. Int J Retin Vitr (2016) 2:3 Page 2 of 9

neovascular age-related (AMD), approximately 15 % glycosylation to give a total molecu- diabetic macular edema (DME) and retinal vein occlu- lar weight of 115 kDa. As a designed molecule featuring sion (RVO)-associated macular edema (ME) [6–8]. These optimal pharmacologic characteristics to inhibit intraoc- retinal diseases are characterized by the production of ular VEGF, intravitreal aflibercept injection (IAI) offers increased levels of intraocular VEGF and development of improved binding affinity and superior pharmacoki- ME resulting in dysfunction of central and sharp vision. netics in an iso-osmotic formulation. Aflibercept may VEGF also mediates the development of neovasculariza- have approximately 100-fold greater binding affinity for tion in these conditions and may lead to severe irreversi- VEGF-A than does either bevacizumab or ranibizumab ble vision loss. The administration of an anti-VEGF agent [9, 10]. It binds to all VEGF-A isoforms and the related in the vitreous cavity of patients with these disorders VEGFR-1 ligands, VEGF-B and PlGF, and it is the only lowers intraocular VEGF, reduces vascular permeabil- United States Food and Drug Administration (FDA)- ity and is associated with arrested growth of and leakage approved VEGF Trap for intravitreal use [9, 14]. from neovessels in choroidal neovascularization (CNV) Ziv-aflibercept (Zaltrap; co-developed by - [6, 9]. Aventis and Regeneron Pharmaceuticals, Inc, Tarrytown, VEGF blockers used to treat eye diseases have included NY, USA), is identical to aflibercept, except for its excipi- an aptamer, a humanized monoclonal , an anti- ents and higher osmolarity. While aflibercept is available body fragment, and, more recently, cytokine traps [6, 10– in a single-use glass vial designed to provide 0.05 ml of 12]. The purpose of this review is to provide an overview 40 mg/ml solution (2 mg) for , ziv- of three fusion proteins that have been studied for the aflibercept is available as 100 mg per 4 ml (25 mg per ml) treatment of retinal diseases: aflibercept, ziv-aflibercept solution or 200 mg per 8 ml (25 mg per ml) solution, in and conbercept. a single-use vial. Aflibercept is iso-osmolar, whereas ziv- aflibercept is hyperosmolar (1000 mOsm/l) relative to the Fusion proteins: history, chemistry, production, vitreous [15, 16]. and biology In 2012, ziv-aflibercept received United States FDA In 2002, Holash et al. published the first paper reporting approval for use in combination with FOLFIRI (folinic the development and in vivo study of VEGF Trap for can- acid, and ) in patients with cer treatment. VEGF Trap was created by fusing the first metastatic colorectal cancer that is resistant to or has three immunoglobulin (Ig) domains of VEGFR-1 to the progressed after -based regimens such as FOL- Fc region of human IgG1. Three additional VEGF traps FOX (, fluorouracil, oxaliplatin) [16]. Its intra- were then engineered on the basis of that initial molecule: vitreal off-label use in humans was not associated with the VEGF Trap B1 (in which a highly basic 10-amino acid toxicity, or higher rate of cataract induction sequence was removed from the third Ig domain of the [11, 17, 18]. Although this drug has a higher osmolarity parental trap), the VEGF Trap B2 (in which the entire when compared to aflibercept, serum and intraocular first Ig domain from VEGF Trap B1 was removed), and osmolarity may not be significantly altered after intravit- VEGF Trap R1R2 (the result of the fusion of the second real injection of ziv-aflibercept [19]. Ig domain of VEGFR-1 with the third domain of VEGFR- In 2008, Zhang et al. published the first study of con- 2). These modifications enhanced R1R2 trap affinity bercept (KH902) (Lumitin; Chengdu Kanghong Bio- for VEGF-A. The initial parental VEGF Trap had very tech, Ltd., Sichuan, People’s Republic of China) in an high affinity for VEGF-A and PlGF, but was a strongly experimental CNV monkey model [20]. Conbercept is positively charged molecule that bound to the extracel- a full human DNA sequence with a molecular weight of lular matrix in addition to VEGF-A and PlGF. Modifica- 143 kDa produced with Chinese hamster ovary cells, and tions resulted in a less positively charged molecule that it combines ligand-binding elements taken from extracel- retained high affinity for VEGF-A and VEGF-B as well lular domain 2 of VEGF receptors 1 (Flt-1) and extracel- as PlGF, but did not specifically bind to the extracellular lular domain 3 and 4 of VEGF receptors 2 (KDR) fused to matrix [13]. the Fc portion of human IgG1 [20–22]. This drug binds The current aflibercept, previously called VEGF Trap VEGF dimers in a 1:1 ratio with a “two-fisted grasp” Eye, evolved from the parental VEGF Trap studied by that resembles the action of aflibercept. The difference Holash et al. This fully human protein consists of an between conbercept and aflibercept is that the former all human amino acid sequence, which minimizes the also contains domain 4 of VEGFR-2, which was proved potential for immunogenicity in human patients [13]. in previous studies to be essential to the receptor [10, 23, Aflibercept (Eylea; Regeneron, Tarrytown, NY, USA, and 24]. Domain 4 does not participate in ligand binding but , Leverkusen, Germany) is a dimeric glycoprotein structure analysis of ligand-bound VEGFR has revealed with a protein molecular weight of 96.9 kDa. It contains that this domain might be involved in specific homotypic de Oliveira Dias et al. Int J Retin Vitr (2016) 2:3 Page 3 of 9

interactions of the ligand-bound receptor, stabilizing receive either 2 mg IAI every 4 weeks (2q4), 2 mg IAI receptor dimers and locking VEGF to the receptor in a every 8 weeks after 5 initial monthly doses (from baseline rigid manner [20]. A preclinical study suggested that to week 16) with sham injections on non-treatment vis- conbercept may have an affinity 50-fold higher for VEGF its (2q8), or macular laser photocoagulation at baseline compared to bevacizumab and that it could be equally and sham injections at every visit [7]. The results dem- more efficient in inhibiting the proliferation of human onstrated that aflibercept given either every 4 or 8 weeks umbilical vein endothelial cells induced by VEGF [20]. was superior to laser alone, and results in both showed Like aflibercept and ziv-aflibercept, conbercept shows significant VA gains and prevention of severe VA loss. strong antiangiogenetic effects by binding with high The mean change from baseline BCVA in the 2q4 and 2q8 affinity and neutralizing VEGF-A, all its isoforms, and groups compared with the laser group was +12.5 ± 9.5 PlGF [12]. and +10.7 ± 8.2 letters vs +0.2 ± 12.5 letters in VISTA Comparative table shows the structural differences (P < 0.0001), and +10.5 ± 9.5 and +10.7 ± 9.3 letters vs between the three fusion proteins available for treatment +1.2 ± 10.6 letters (P < 0.0001) in VIVID, respectively of retinal diseases (Table 1). [7]. The percentage of eyes in the laser group that lost ≥15 letters of vision replicated the 10 % loss reported by Clinical application of fusion proteins the early treatment study (ETDRS) in ophthalmology for treatment of retinal diseases [7, 28]. Overall incidences of ocular and nonocular AE Aflibercept were similar across treatment groups [7]. The United States FDA approved Aflibercept in Novem- The Diabetic Retinopathy Clinical Research Network ber 2011 for the treatment of neovascular AMD, in Octo- (DRCR.net), sponsored by the National Institutes of ber 2014 for ME following RVO, and in March 2015 for Health, conducted a multicenter, randomized clinical the treatment of DME [9, 14, 25–27]. trial at 89 clinical sites in the United States to compare To date, large phase 3 studies have been conducted to the efficacy and safety of intravitreous aflibercept, bevaci- evaluate the efficacy and safety of intravitreal aflibercept zumab, and ranibizumab for the treatment of DME caus- for exudative AMD. The VEGF Trap-Eye: Investigation of ing decreased VA. The study drugs were injected into efficacy and safety in wet AMD (VIEW 1 and 2) recruited the study eyes at baseline and then every 4 weeks unless treatment-naive patients with neovascular AMD from VA was 20/20 or better with a central subfield thickness 362 centers worldwide [14]. From baseline to week 52, below the eligibility threshold and there was no improve- patients received 0.5 mg intravitreal ranibizumab every ment or worsening in response to the past two injections. 4 weeks (Rq4), 2 mg aflibercept every 4 weeks (2q4), Laser photocoagulation (focal, grid, or both) was 0.5 mg aflibercept every 4 weeks (0.5q4), or 2 mg afliber- initiated at or after the 24-week visit for persistent DME. cept every 8 weeks (2q8) after 3 monthly injections. Between August 2012, and August 2013, 660 participants During weeks 52 through 96, patients received their were randomly assigned to receive aflibercept 2.0 mg original dosing assignment using an as-needed regimen (224 participants), bevacizumab 1.25 mg (218), or ranibi- with defined retreatment criteria and mandatory dosing zumab 0.3 mg (218). The median number of injections at least every 12 weeks. All aflibercept and ranibizumab was 9 or 10 in the three groups. The mean improvement groups were equally effective in improving best-cor- in the VA letter score at 1 year was greater with afliber- rected visual acuity (BCVA) and maintaining BCVA (lost cept than with bevacizumab or ranibizumab (13.3 vs 9.7 <15 letters from baseline) at 96 weeks. The 2q8 afliber- and 11.2, respectively; P < 0.001 for aflibercept vs bevaci- cept group was similar to ranibizumab in visual acuity zumab and P = 0.03 for aflibercept vs ranibizumab), but (VA) outcomes during 96 weeks, but with an average of the relative effect varied according to initial VA. At the 5 fewer injections [14]. Over the 2 years of treatment, a 1-year visit, the central subfield thickness decreased, on generally favorable safety profile was observed for both average, by 169 ± 138 μm with aflibercept, 101 ± 121 μm intravitreal aflibercept and ranibizumab. The incidence with bevacizumab, and 147 ± 134 μm with ranibizumab. of ocular treatment-emergent adverse events (AE) was Injection-related infectious endophthalmitis occurred in balanced across all treatment groups, with the most fre- one aflibercept-treated eye and one ranibizumab-treated quent events associated with the injection procedure, eye (both nonstudy eyes) and no bevacizumab-treated the underlying disease, the aging process, or a combina- eyes. Through 1 year, the rate of serious adverse events tion thereof. The incidences of arterial thromboembolic was similar in the three treatment groups (P = 0.40), as events and death were similar across all treatment groups was the rate of hospitalization (P = 0.51) [29]. [14]. Intravitreal aflibercept has also been investigated In two parallel phase 3 DME studies, VISTADME and for the treatment of ME secondary to central reti- VIVIDDME, eyes were randomized in a 1:1:1 ratio to nal vein occlusion (CRVO) in two parallel trials, the de Oliveira Dias et al. Int J Retin Vitr (2016) 2:3 Page 4 of 9 PIGF and fourth domains of VEGFR2 to the Fc portion of the Fc VEGFR2 to and fourth domains of human IgG1 days (rabbits) 4, 2 days Not published 143 kDa Kd 0,5 Pm 10 mg/ml Binds with all isoforms of VEGF-A, VEGF-B, VEGF-C and VEGF-B, VEGF-A, of Binds with all isoforms Fusion of the second domain of VEGFR1 and the third VEGFR1 and the third of the second domain Fusion Conbercept third domain of VEGFR 2 to the Fc portion of the Fc VEGFR 2 to domain of third human IgG1 7, 1 days 1000 mOsm 115 kDa Kd 0,49 Pm 25 mg/ml Binds to all isoforms of VEGF-A, VEGF-B and PIGF VEGF-A, of all isoforms Binds to Fusion of the second domain of VEGFRs 1 and the of the second domain Fusion Ziv-aflibercept third domain of VEGFR 2 to the Fc portion of the Fc VEGFR 2 to domain of third human IgG1 7, 1 days 286 mOsm 115 kDa Kd 0,49 Pm 40 mg/ml Binds to all isoforms of VEGF-A, VEGF-B and PIGF VEGF-A, of all isoforms Binds to Fusion of the second domain of VEGFRs 1 and the of the second domain Fusion Aflibercept Comparative table showing the structural differences between the three fusion proteins available for treatment of retinal diseases of retinal for treatment available fusion proteins three between the the structural differences table showing Comparative Half-life Osmolarity Molecular weight Binding affinity to VEGF-A (165) Binding affinity to Concentration Mechanism of action Structure 1 Table de Oliveira Dias et al. Int J Retin Vitr (2016) 2:3 Page 5 of 9

COPERNICUS and GALILEO studies, performed in the at 24 weeks than did grid laser. The proportion of eyes United States and in Europe and Asia/Pacific, respec- that gained ≥ 15 ETDRS letters from baseline at week 24 tively [8, 30, 31]. In the GALILEO study, patients were was 52.7 % in the IAI group compared with 26.7 % in the randomized to receive either 2 mg IAI or sham in the laser group (P = 0.0003). The mean improvement from study eye once every 4 weeks for 20 weeks, for a total of baseline BCVA at week 24 was 17.0 ETDRS letters in six doses. From week 24 to week 48, patients in the IAI the IAI group and 6.9 ETDRS letters in the laser group group were evaluated every 4 weeks and received afliber- (P < 0.0001). The mean reduction in CRT from baseline cept as needed or pro re nata (PRN) if they met prespeci- at week 24 was 280.5 µm in the IAI group and 128.0 µm fied retreatment criteria [8]. The proportion of patients in the laser group (P < 0.0001). Traumatic cataract in an who gained ≥ 15 letters in the intravitreal aflibercept and IAI patient was the only ocular SAE that occurred. The sham groups was 60.2 vs 22.1 % at week 24 (P < 0.0001), incidence of nonocular SAE was 8.8 % in the IAI group 60.2 vs 32.4 % at week 52 (P < 0.001), and 57.3 vs 29.4 % and 9.8 % in the laser group [25]. at week 76 (P < 0.001). The COPERNICUS study was a trial parallel to GALILEO, differing in the timing of the Ziv‑aflibercept IAI. Patients received IAI 2 mg (IAI 2q4) or sham injec- Ziv-aflibercept received FDA approval in August 2012 tions every 4 weeks up to week 24. During weeks 24 to for the treatment of metastatic colorectal carcinoma that 52, patients from both arms were evaluated monthly is resistant to or has progressed on an oxaliplatin-based and received IAI PRN (IAI 2q4 + PRN and sham + IAI regimen [16]. Due to its mechanism of action, there is PRN). During weeks 52 to 100, patients were evaluated at growing interest in using intravitreal ziv-aflibercept as an least quarterly and received IAI PRN. The most frequent antiangiogenic agent for ophthalmic VEGF-related dis- ocular severe adverse event (SAE) from baseline to week eases [17]. 100 was vitreous hemorrhage (0.9 vs 6.8 % in the IAI Malik et al. exposed human RPE cells for 24 h to four 2q4 + PRN and sham + IAI PRN groups, respectively) [8, anti-VEGF drugs (bevacizumab, ranibizumab, aflibercept 30, 31]. Overall, the COPERNICUS study demonstrated and ziv-aflibercept) at 1/2, 1, 2 and 10× clinical concen- similar effects as those seen in GALILEO study in visual tration. At clinical doses (1×) there was no decrease in and anatomic improvements with IAIs after switching cell viability in all four drug groups [32]. In an experi- from monthly dosing [30]. mental study, nine rabbits were given an intravitreal The VIBRANT study was conducted to compare the injection of 0.05 ml ziv-aflibercept (25 mg/ml) [19]. There efficacy and safety of IAI with macular grid laser pho- were no associated complications such as cataract and tocoagulation for the treatment of ME after branch retinal detachment (RD). All eyes showed no signs of retinal vein occlusion (BRVO) [25]. It showed that toxicity on funduscopy, optical coherence tomography monthly IAI provided significantly greater visual ben- (OCT) (Fig. 1), and full-field electroretinogram (ERG) 1 efit and reduction in central retinal thickness (CRT) or 7 days after the procedure. There were also no changes

Fig. 1 Spectral-domain OCT 7 days after intravitreal injection of 0.05 mL of ziv-aflibercept (25 mg/mL) (a) or aflibercept (40 mg/mL) (b) in two rab- bits’ right eye de Oliveira Dias et al. Int J Retin Vitr (2016) 2:3 Page 6 of 9

in median baseline serum, vitreous, and aqueous osmo- usual volume of 0.05 ml for intravitreal injections. Vol- larities. Histology and transmission electron microscopy umes larger than 0.05 ml may increase the risk of intraoc- showed no major anatomic signs of toxicity, and no cyto- ular pressure [11]. Unpublished data from the DME study toxic effect was observed in ARPE-19 cells exposed to group from the Federal University of São Paulo showed clinical and 2× clinical concentration of ziv-aflibercept, regression of intraretinal fluid in a 49-year-old female which was also reported by Bababeygy et al. [19, 33]. del patient presenting DME, 4 weeks after the third injec- Carpio et al. showed that ziv-aflibercept was not detri- tion of 0.05 ml of ziv-aflibercept 25 mg/ml (1.25 mg) mental to cell viability at a low dose (1/2×) and a clini- (Figs. 2 and 3 a, b, c). VA improved from 20/100 to 20/40, cal equivalent dose for MIO-M1 cells in vitro. However, 4 weeks after the third injection of ziv-aflibercept. twice the clini-cal dose of ziv-aflibercept reduced Müller Mansour et al. presented recent data on four patients cell viability, whereas this reduction at 2× clinical dose with neovascular AMD and two patients with DME was not observed for ranibizumab, aflibercept, or beva- who received one intravitreal injection of 0.05 ml of cizumab [34]. ziv-aflibercept (1.25 mg). All six patients showed evi- Recently, de Oliveira Dias et al. reported the first dence of improvement in VA with no signs of intraocu- human intravitreal administration of ziv-aflibercept. A lar inflammation, change in lens status or retinal toxicity patient with refractory neovascular AMD was given 2 [17]. Chhablani also reported the intravitreal injection of monthly intravitreal injections of 0.05 ml ziv-afliber- 1.25 mg intravitreal ziv-aflibercept in a patient presenting cept (1.25 mg) and experienced subjective and objective with bilateral ME secondary to CRVO, who had already improvement in VA with a decrease in intraretinal and received 12 and 13 anti-VEGF injections in the right and subretinal fluid. No ERG changes were noticed when left eye, respectively, along with one intravitreal triamci- baseline and 30-day follow-up were compared. No AE nolone injection and peripheral panretinal photocoagu- were observed at any time point. The therapeutic dose lation in both eyes. At 1-month follow-up of intravitreal of ziv-aflibercept was chosen on the basis of preliminary ziv-aflibercept injection in both eyes, the patient experi- studies that showed efficacy of aflibercept at doses of enced an improvement in VA along with CRT decrease. 0.5–2 mg. The dose of 1.25 mg was chosen to keep the He did not have any symptoms of blurred vision or ocular

Fig. 2 Baseline color fundus image (a), fluorescein angiography (b) and SD-OCT (c) of the right eye of a patient presenting DME. a At baseline, hard exudates and diffuse intraretinal fluid are seen in the perifoveal area. b At baseline, diffuse hyperfluorescence due to leakage (especially supero- temporally) and hypofluorescence due to non-perfusion (infero-temporally) are seen in the perifoveal area. c Cystoid spaces and subretinal and intraretinal fluid are seen in the foveal and perifoveal area de Oliveira Dias et al. Int J Retin Vitr (2016) 2:3 Page 7 of 9

Fig. 3 Color fundus image (a) fluorescein angiography (b) and SD-OCT (c) of the right eye of the same patient shown in Fig. 2. 4 weeks after the 3rd monthly injection of ziv-aflibercept. a 4 weeks after the third ziv-aflibercept injection, a decrease of intraretinal fluid and hard exudates is noticed in the perifoveal area of the right eye. b 4 weeks after the third ziv-aflibercept injection, a decrease of leakage is seen in the perifoveal area of the right eye. c 4 weeks after the third ziv-aflibercept injection, a decrease in the subretinal and intraretinal fluid is noticed

pain related to injection or any signs of inflammation/ and 2.0-mg Q1 M regimens, respectively. A reduction in toxicity [35]. CRT was also detected. At 12 months, mean reductions in CRT in the four regimens were 119.8, 129.7, 152.1, and Conbercept 170.8 μm, respectively. At 12 months follow-up, no sig- Conbercept (KH902) has been studied in a phase 3 clini- nificant differences in BCVA or anatomic outcomes were cal trial and was approved to treat neovascular AMD by found between the groups, regardless of the dose or dos- the China State FDA in December 2013. Conbercept has ing regimen. Overall, conbercept was well tolerated, and not yet reached the market in other countries [36]. the incidence of ocular AE was low. The most common After preclinical results showing the antiangiogenetic AE were usually caused by the intravitreal injection pro- effects of conbercept, there has been a growing interest cedure and disappeared with or without treatment. RD in using this drug intravitreally to treat VEGF-related did not occur in this study. A case of hepatitis suspected ophthalmic diseases [12, 20, 36]. The AURORA study was of being drug induced was identified and thought to be a 12-month, randomized, double-masked, controlled- caused by an oral supplement, and the hepatitis was cat- dose, and interval-ranging phase two clinical trial that egorized as having no relationship with the study drug took place at nine sites in China; it was designed as a [12]. superiority trial to assess the safety and efficacy of differ- A phase 3 trial with intravitreal conbercept for exuda- ent dosing regimens. Eligible patients were randomized tive AMD was completed in 2013, and the results were 1:1 to 0.5- or 2.0-mg treatment groups. Initially, all announced at Angiogenesis, Exudation, and Degenera- patients received monthly intravitreal injections of con- tion in 2014 [37, 38]. In this trial, patients in the treat- bercept for a total of three injections. After three loading ment group received three fixed monthly injections and doses of monthly intravitreal conbercept injection, the then two sham injections monthly followed by conber- patients were then randomized into the monthly (Q1 M) cept injection every 3 months up to 12 months (intravit- or PRN group. One hundred and twenty-two patients real con-bercept at 0, 1, 2, 5, 8, and 11 months). Patients were enrolled. At 12 months, mean improvements in in the sham injection group were given three monthly BCVA from baseline were 14.31, 9.31, 12.42, and 15.43 sham injections and then crossed over to the treat- letters for the 0.5-mg PRN, 0.5-mg Q1 M, 2.0-mg PRN, ment group. Both groups had a 12-month follow-up de Oliveira Dias et al. Int J Retin Vitr (2016) 2:3 Page 8 of 9

period. The report from the Angiogenesis, Exudation, revised the paper. MEF wrote the review and revised the paper. EBR advised the writing of the manuscript, revised. All authors read and approved the final and Degeneration meeting in 2014 showed that patients manuscript. exhibited a mean change in BCVA of +10 letters at 12 months. Morphologic changes observed by OCT Acknowledgements exhibited a significant reduction in central subfield Publication of this article was supported by Fundação de Amparo à Pesquisa thickness of 79 μm in the treatment groups at 3 months, do Estado de São Paulo (FAPESP; São Paulo, Brazil), Conselho Nacional de whereas the sham injection group experienced a Desenvolvimento Científico e Tecnológico (CNPq; Brasília, Brazil), and Pan- American Association of Ophthalmology/Pan-American Ophthalmological decrease of 44 μm, which was not significant. When Foundation, Paul Kayser Global Award (PAAO/PAOF; Arlington, Texas). the sham injection group crossed over to the treatment group, similar results were also observed at 12 months Competing interests The authors declare that they have no competing interests. on OCT and fluorescein angiography. A reduction in subretinal fluid maximum height at 3 months was seen Received: 20 March 2015 Accepted: 26 November 2015 in the treatment group. After crossing over to the treat- ment group, 93 % of participants had less than 320 μm central subfield thickness at 12 months [20, 37, 38]. References Conclusions 1. Ferrara N, Davis-Smyth T. The biology of vascular endothelial growth fac- Fusion proteins are a promising treatment for ocular dis- tor. Endocr Rev. 1997;18:4–25. 2. Zachary I. Vascular endothelial growth factor and anti-angiogenic pep- eases related to angiogenesis. They are fully human and tides as therapeutic and investigational molecules. 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